The severity and duration of food-borne diseases are influenced by plasmid-encoded virulence factors and antibiotic resistance genes. Environmental pathogens such as Escherchia coli, Salmonella, and Shigella, which are transmitted to the food chain from polluted irrigation and fertilization systems, have been shown to possess both of these plasmid-encoded elements. Patients infected with either virulent or antibiotic resistant microorganisms from contaminated food face severely limited treatment options or significantly prolonged therapeutic regimens. My long term objectives are to increase our understanding of conjugative plasmid transfer between environmental pathogens as it relates to the spread of antibiotic resistance genes and virulence factors. Conjugative plasmid transfer is the method by which these elements are spread among pathogenic and non-pathogenic microbial strains. Transfer of the prototypical F (fertility) plasmid and the numerous R (resistance) plasmids is mediated by a plasmid-specific Tral enzyme. We have identified potent (EC50 and Ki values in the nanomolar range) inhibitors of F plasmid transfer and Tral-mediated plasmid cleavage. Because F and R plasmid Tral enzymes share up to 98% sequence identity, I hypothesize that similar small molecules will be effective inhibitors of R plasmid transfer. The specific aims of this proposal are to: first, identify potent inhibitors of the F plasmid Tral enzyme; second, compare the structure of the Tral enzyme from the F plasmid to those of R plasmid Tral enzymes; third, identify inhibitors targeting the R plasmid Tral enzymes. I will use a combination of molecular biology and biochemistry to elucidate the structural basis of Tral inhibition and to identify new inhibitors of Tral enzymes encoded on the both F and R plasmids. This is a necessary first step to address the widespread and growing problem of antibiotic resistance.